InteractiveFly: GeneBrief

Sas-4: Biological Overview | References

Centrioles and centrosomes have an important role in animal cell organization, but it is uncertain to what extent they are essential for animal development. The Drosophila protein Sas-4 is related to the human microcephaly protein CenpJ and the C. elegans centriolar protein Sas-4 (Leidel, 2003; Kirkham, 2003). Drosophila Sas-4 is essential for centriole replication in flies. Sas-4 mutants start to lose centrioles during embryonic development, and, by third-instar larval stages, no centrioles or centrosomes are detectable. Mitotic spindle assembly is slow in mutant cells, and 30% of the asymmetric divisions of larval neuroblasts are abnormal. Nevertheless, mutant flies develop with near normal timing into morphologically normal adults. These flies, however, have no cilia or flagella and die shortly after birth because their sensory neurons lack cilia. Thus, centrioles are essential for the formation of centrosomes, cilia, and flagella, but, remarkably, they are not essential for most aspects of Drosophila development (Basto, 2006)

Since their first description more than 100 years ago, centrosomes have been recognized as important organizers of animal cells (see The centrosome cycle in mammalian cells from Azimzadeh, 2007 from Azimzadeh, 2007). They consist of a pair of centrioles surrounded by an amorphous pericentriolar material (PCM), which nucleates and organizes microtubules (MTs). Through the MTs they organize, centrosomes are thought to have important roles in establishing cell polarity, positioning organelles within cells, directing intracellular traffic, and organizing cell division (Basto, 2006).

Although centrosomes are major organizers of animal cell division, they are not essential for mitotic spindle assembly. Some animal cells normally organize their spindles without canonical centrosomes, and cultured cells that have had their centrosomes removed by laser ablation or microsurgery can still form bipolar spindles. In these cases, the mitotic chromosomes appear to initiate the assembly of a bipolar spindle and thereby compensate for the lack of centrosomes. Acentrosomal cultured cells, however, often fail in cytokinesis, and, even if these cells complete cytokinesis successfully, they usually arrest in the following G1 phase. This has led to the suggestion that cells have a checkpoint that monitors centrosome integrity in G1 (Basto, 2006)

Although centrosomes are dispensable for spindle assembly in many cell types, it is widely believed that they are essential for asymmetrical division because astral MTs directly contact cues in the cell cortex to position the mitotic spindle appropriately within the cell. Studies of centrosomin (cnn) and asterless (asl) mutants in Drosophila, however, suggest that centrosomes and astral MTs may not be essential for the asymmetric divisions of larval neuroblasts (Bonaccorsi, 2000; Giansanti, 2001; Megraw, 2001). These mutants appear to lack functional mitotic centrosomes, yet their neuroblasts have only subtle defects in aligning their spindles with cortical cues during early mitosis, and, by telophase, almost all neuroblasts appear to divide asymmetrically, just as in wild-type (wt) larvae. It remains controversial, however, whether cnn and asl mutants completely lack functional mitotic centrosomes (Basto, 2006)

Consistent with their many functions, centrosome dysfunction has been implicated in a wide variety of human diseases. Centrosome defects are believed to contribute to the genetic instability associated with many cancers, and genetic studies have implicated centrosomes in microcephaly, a condition associated with a small brain size at birth. Of the four genes so far linked to microcephaly, three (ASPM, Cdk5Rap2, and CenpJ) encode centrosomal proteins (Bond, 2005; Kouprina, 2005). It has been postulated that the small brain size in these individuals may be caused by defects in asymmetric division in the neural precursor cells that generate neurons during early fetal development (Woods, 2005). In addition, the centrioles in many animal cells are thought to have important functions that are distinct from their function as organizers of the centrosome. They form the basal bodies that nucleate the formation of cilia and flagella, and cilia defects contribute to a variety of human diseases (Basto, 2006)

Despite the likely importance of centrioles and centrosomes in so many cell processes, few experiments have addressed whether they have essential roles during animal development. The polo-like kinase Plk-4/Sak is essential for centriole replication (Bettencourt-Dias, 2005; Habedanck, 2005), and Drosophila sak hypomorphic mutant third-instar larval brains appear to lack centrioles in ~20% of their cells, yet they develop into adults at rates that are only slightly slower than normal (Bettencourt-Dias, 2005). The centrioles that are present in sak mutants, however, appear to be fully functional, so it is difficult to infer whether centrioles or centrosomes have essential roles in Drosophila development. Centrioles and centrosomes are essential for the development of C. elegans embryos. Five C. elegans proteins, Sas-4, Sas-5, Sas-6 (Leidel, 2003; Leidel, 2005a), Spd-2 (see Drosophila Spd-2), and Zyg-1 (the likely homolog of Plk-4/Sak) are required for centriole replication in worm embryos, and perturbing the function of any of them arrests embryonic development at the one or two cell stage. This early arrest, however, precludes an analysis of the importance of centrioles and centrosomes at later stages of worm development. This study show that a mutation in the Drosophila Sas-4 gene shows that centrioles and centrosomes are not essential for most aspects of Drosophila development (Basto, 2006)

The Drosophila protein encoded by the gene CG10061 shares a C-terminal domain with the human centrosomal protein CPAP/CenpJ and an N-terminal domain with the C. elegans centriolar protein Sas-4. CG10061 appears to function in a similar manner to C. elegans Sas-4, and is there for referred to as Drosophila Sas-4 (Basto, 2006)

Antibodies were raised against the N-terminal region of Sas-4. Affinity-purified anti-Sas-4 antibodies recognized a small dot at the center of the centrosome at all stages of the cell cycle in both embryos and larval neuroblasts. This staining was absent in Sas-4 mutant larval neuroblasts. Such dot-like centrosomal staining is usually indicative of centriole staining, and Sas-4 colocalizes with the Drosophila centriole markers GFP-PACT and GTU88^* (Martinez-Campos, 2004) in fixed larval neuroblasts. Moreover, anti-Sas-4 antibodies stain the very large centrioles found in spermatocytes; this staining is absent in Sas-4 mutant spermatocytes. Thus, Sas-4 is closely associated with centrioles (Basto, 2006)

Sas-4 mutant cells progressively lose centrioles during embryonic development as the maternally supplied Sas-4 protein is exhausted; by first-instar larval stages, ~90% of mutant brain cells lack detectable centrioles, and by third-instar larval stages, centrioles are essentially undetectable in these cells. No centrosomes, cilia, or flagella are detected in mutant cells that lack centrioles, strongly suggesting that centrioles are essential for the formation of these structures in flies. Remarkably, these mutant flies develop at near-normal rates and are born at near normal Mendelian ratios, demonstrating that flies can proceed through the majority of development without centrioles, centrosomes, cilia, or flagella. Mutant adults, however, die shortly after birth because they lack cilia in type I sensory neurons. Thus, centrioles are essential for fly survival only because they are required for cilia formation (Basto, 2006)

In C. elegans, centrioles and centrosomes are essential for early development: mutant embryos that cannot replicate their centrioles arrest after only one or two rounds of cell division. The same would probably be true for the earliest stages of Drosophila embryogenesis, as one would expect centrosomes to be especially important in early syncytial Drosophila embryos, in which hundreds of large spindles have to assemble and disassemble very quickly within a common cytoplasm. In Sas-4 mutants, however, the heterozygous mothers contribute Sas-4 to the early embryos, which therefore contain centrioles and centrosomes (Basto, 2006)

It is clear from previous studies that centrosomes are not required for spindle assembly, since mitotic chromosomes and MT-dependent motor proteins can organize the assembly of bipolar spindles. It is nonetheless surprising that centrioles and centrosomes are dispensable for cell division during most stages of Drosophila development. Although a Drosophila cell line that lacks centrioles has been identified, these cells often fail to divide normally. Cultured mammalian cells that have had their centrosomes removed also often fail to complete cytokinesis, and those cells that do divide often then arrest in G1 of the next cycle, suggesting that centrosomes are required for both efficient cytokinesis and cell-cycle progression. One might expect, therefore, that an animal lacking centrosomes would, at the very least, be at a severe growth disadvantage compared to a normal animal. This seems not to be the case in Drosophila. Although spindle assembly is slowed in acentrosomal Sas-4 mutant cells, once assembled, these spindles make few chromosome-segregation errors. Moreover, the ~30%-40% increase in the duration of mitosis in mutant cells does not significantly delay development, probably because mitosis occupies such a small fraction of the total cell cycle. Thus, in flies at least, centrioles and centrosomes are not essential for any aspect of somatic cell-cycle progression or cell division; unlike cultured mammalian cells, fly cells do not arrest in G1 if they have no centrioles or centrosomes (Basto, 2006)

Centrioles or centrosomes were found to have an important role in asymmetric cell division in Drosophila. This contrasts with previous studies on cnn and asl mutants, which also appear to lack functional mitotic centrosomes. The cortical cues that guide asymmetric division are localized normally in these mutants, although spindles fail to align efficiently with these cues at early stages of mitosis. By telophase, however, ~90% of the mutant cells have properly aligned spindles, and the cells appear to divide asymmetrically. In Sas-4 mutants, by contrast, at least two aspects of asymmetric division are perturbed. (1) The localization of Miranda to a basal cortical crescent occurs unreliably, suggesting that centrosomes play an important part in establishing and/or maintaining cortical Miranda during asymmetric division. (2) ~30% of mutant neuroblasts either divide symmetrically or fail to complete cytokinesis. It is suspected that the explanation for the differences between the Sas-4 mutants and the cnn and asl mutants is that the centrosomes in cnn and asl mutants are partially functional, whereas they are completely absent in Sas-4 mutants. Cnn mutants, for example, have centrioles, and astral MTs can be detected in at least some cnn mutant neuroblasts using live-cell imaging techniques, which are more sensitive than those used in previous studies (Basto, 2006)

These observations fit well with recent evidence for two partially redundant mechanisms that ensure the fidelity of asymmetric division in embryonic fly neuroblasts. The first mechanism is MT independent and is initiated prior to the entry into mitosis by Insc/Par protein complexes concentrated at the apical cortex. These complexes recruit Pins/Gαi complexes, which then help drive the redistribution of proteins like Miranda to the basal cortex. In the absence of Insc/Par complexes, a MT-dependent mechanism can recruit Pins/Gαi complexes to a cortical region adjacent to one of the spindle poles. Presumably, these two mechanisms normally cooperate to ensure that the forming spindle efficiently aligns with preexisting cortical cues. If one mechanism is perturbed, however, the other is apparently sufficient to allow neuroblasts to divide asymmetrically. This redundancy presumably explains why neuroblasts carrying mutations that affect asymmetric division usually have misaligned spindles at metaphase but are “rescued” by telophase and so ultimately divide asymmetrically. The current analysis emphasizes that these two mechanisms are only partially redundant: Miranda does not consistently localize to the basal cortex in Sas-4 mutant cells, even though the Insc/Par complex almost always localizes correctly. Moreover, telophase rescue is inefficient in these cells, and ~30% of cells either fail in cytokinesis or divide symmetrically. Thus, Drosophila neuroblasts apparently have great difficulty in compensating for a lack of centrosomes (Basto, 2006)

Despite these difficulties, ~70% of acentrosomal Sas-4 mutant larval neuroblasts divide asymmetrically. How can cells that lack centrosomes and astral MTs align their spindle with cortical cues so as to divide asymmetrically? Live-cell analysis provides a potential explanation. Many acentrosomal spindles extend across the full length of the cell, so that the spindle poles are in close contact with the cortex, and this was also a noticeable feature of the mutant spindles analyzed by EM. This may allow the acentrosomal spindles to interact directly with cortical cues even in the absence of astral MTs, perhaps explaining how the majority of these spindles ultimately align correctly (Basto, 2006)

It is even more surprising that flies in which ~30% of the brain neuroblasts fail to divide properly seem to have so few developmental defects. The brain seems grossly normal in size, morphology, and histology. Moreover, the neuronal axons in the developing eye disc seem to be oriented correctly, which is unexpected, since previous studies have suggested that the initial direction of axon outgrowth in these cells is defined by the position of the centrosome (de Anda, 2005). DSas-4 mutants may well have subtle defects in neuronal development such as mild proliferation defects which would require lineage trancing experiments to be detected. However, it is clear that the developing fly brain has a remarkable ability to compensate for large-scale abnormalities in neuroblast divisions. It is unclear how the brain manages this. In humans, mutations in CenpJ, the homolog of DSas-4, results in microcephaly, which has been proposed to be caused by abnormalities in neural precursor cell divisions during fetal development (Woods, 2005). The finding that neuroblast divisions are frequently abnormal in Sas-4 mutant flies provides the first direct support for this proposal, although the brains of Sas-4 mutants appear no smaller than wt brains. Perhaps the developing human brain cannot compensate for abnormalities in neural precursor cell divisions in the way that the developing fly brain can (Basto, 2006)

It will be of great interest to determine whether centrioles and centrosomes are largely dispensable for much of development in other organisms. This may be difficult to address in other systems. In flies, only type I mechanosensory neurons and sperm have cilia and flagella, respectively, so the lack of centrioles produces only an uncoordinated phenotype. By contrast, many types of vertebrate cells have a primary cilium, which, in some cells at least, is required for the cell to respond to certain extracellular signals. Moreover, cilia in vertebrates have crucial roles in the development of organs such as the kidney. Thus, a lack of centrioles is likely to have a more devastating effect on vertebrate development than on fly development, which might make it difficult to assess whether developing vertebrates can compensate for the lack of centrioles and centrosomes in cell division in the way that Drosophila apparently can (Basto, 2006)

Centrosomes are thought to influence many aspects of cell behavior, including cell migration and cell polarity. These findings suggest that centrosomes are not essential for any of these processes during most of fly development. It remains unclear, however, whether these processes do not depend on centrosomes in Drosophila or whether they are normally dependent but can compensate for the absence of centrosomes. Sas-4 mutants should provide a useful model to explore the importance of centrosomes in many cell processes (Basto, 2006)

Sequential protein recruitment in C. elegans centriole formation

Formation of the microtubule-based centriole is a poorly understood process that is crucial for duplication of the centrosome, the principal microtubule-organizing center of animal cells. Five proteins have been identified as being essential for centriole formation in C. elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6, and SPD-2. The relationship between these proteins is incompletely understood, limiting understanding of how they contribute to centriole formation. This study established the order in which these five proteins are recruited to centrioles, and molecular epistasis experiments were conducted. SPD-2 is loaded first and is needed for the centriolar localization of the four other proteins. ZYG-1 recruitment is required thereafter for the remaining three proteins to localize to centrioles. SAS-5 and SAS-6 are recruited next and are needed for the presence of SAS-4, which is incorporated last. These results indicate in addition that the presence of SAS-5 and SAS-6 allows diminution of centriolar ZYG-1. Moreover, astral microtubules appear dispensable for the centriolar recruitment of all five proteins. Several of these proteins have homologs in other metazoans, and it is expected that the assembly pathway that stems from this work is conserved (Delattre, 2006).

Centrioles are minute cylindrical structures that contain nine sets of microtubules arranged in a radial fashion. At the onset of the centrosome duplication cycle, the two tightly apposed centrioles split slightly from one another. Each of these mother centrioles then seeds formation of a daughter centriole. Centriole formation has been described by ultrastructural analysis in vertebrate cells that primarily monitored the growth of the microtubules, which constitute the defining feature of centrioles. By contrast, the molecular tenets of this assembly process have remained elusive (Delattre, 2006).

The C. elegans embryo has proven well suited for investigating centrosome duplication. The five proteins known to be essential for centriole formation in this organism are enriched at centrioles and present at lower levels in the cytoplasm of early embryos. ZYG-1 is found at centrioles primarily during anaphase, whereas the four other proteins are centriolar throughout the cell cycle. Furthermore, SPD-2 is enriched in the PCM compared to the cytoplasm, possibly reflecting its additional role in PCM assembly (Delattre, 2006).

The order in which ZYG-1, SAS-4, SAS-5, SAS-6, and SPD-2 are recruited to centrioles was investigated. Experiments were designed that distinguish de novo centriolar recruitment from the prior presence of proteins at centrioles. Such experiments are rendered possible because the sperm contributes the sole pair of centrioles to the newly fertilized embryo. These two centrioles split slightly from one another, and each seeds the formation of a daughter centriole. Because the initial pair of centrioles is of paternal origin, one can assay specifically centriolar recruitment or exchange that occurs in the one-cell-stage embryo, provided the centrioles contributed by the sperm do not harbor the protein under scrutiny (Delattre, 2006).

Initially, SPD-2 was analyzed. Because GFP-SPD-2 is not present in sperm, in contrast to the endogenous protein, the time at which GFP-SPD-2 is first detected at centrioles was determined in one-cell-stage embryos. Double labeling was used with antibodies against SAS-4 to mark all centrioles and against GFP to detect GFP-SPD-2 recruitment. GFP-SPD-2 was first detected at centrioles during meiosis I. Even though endogenous SPD-2 is present in sperm, it is lost rapidly after fertilization in embryos depleted of SPD-2. Therefore, recruitment of the endogenous protein was studied and it was found that, as for GFP-SPD-2, SPD-2 is first detected at centrioles during meiosis I. Endogenous ZYG-1 is not present in sperm, which enabled assay of its recruitment after fertilization. ZYG-1 was also first detected at centrioles during meiosis I. In conducting these experiments, it was noted that the paternally contributed centrioles can be first distinguished a single entities during meiosis II, indicating that splitting has occurred by that time. Overall, it is concluded that SPD-2 and ZYG-1 centriolar recruitment initiates as early as meiosis I, prior to when splitting of the centriole pair can be observed by light microscopy (Delattre, 2006).

Next, SAS-5 was examined. In this case, both the endogenous protein and GFP-SAS-5 are present in sperm centrioles. Therefore, marked mating experiments were performed by crossing hermaphrodites expressing GFP-SAS-5 to wild-type males, the sperm of which provide centrioles not carrying the fusion protein. By contrast to the situation with SPD-2 and ZYG-1, it was found that GFP-SAS-5 is not present at centrioles during meiosis I. Instead, GFP-SAS-5 is first detected weakly at the end of meiosis II, with the centriolar signal becoming more robust thereafter. Because GFP-SAS-6 is not present in sperm, in contrast to the endogenous protein, it was possible to assess when the fusion protein is first recruited to centrioles after fertilization. It was found that centriolar GFP-SAS-6 is first detected shortly after meiosis II and more robustly soon thereafter, much like GFP-SAS-5. This is in line with the fact that SAS-5 and SAS-6 physically interact and are mutually dependent for their centriolar localization. Overall, it is concluded that SAS-5 and SAS-6 are recruited after SPD-2 and ZYG-1 (Delattre, 2006).

Because both SAS-4 and GFP-SAS-4 are present in sperm centrioles 3 and 4, marked mating experiments were conducted to investigate GFP-SAS-4 centriolar recruitment in one-cell-stage embryos. GFP-SAS-4 was incorporated progressively to centrioles during the first cell cycle, starting at the time of pronuclear formation. Taken together, these observations establish the following temporal sequence of recruitment to centrioles: first, SPD-2 and ZYG-1; second, SAS-5 and SAS-6; and third, SAS-4 (Delattre, 2006).

Next, whether this temporal sequence corresponds to related episatic interactions was assessed. In one extreme scenario, the five proteins could be recruited independently of one another. Alternatively, the proteins that are recruited early in the sequence may be needed for the presence of some that are recruited later. Whether SPD-2 is required for the centriolar recruitment of the other four proteins was investigated.ZYG-1, GFP-SAS-5, GFP-SAS-6, and GFP-SAS-4 all fail to be recruited to centrioles in spd-2(RNAi) embryos. Moreover, levels of SAS-4 on paternally contributed centrioles are diminished in spd-2(RNAi) embryos compared to the wild-type, as suggested by previous observations. Because SAS-4 is stably associated with the centriole in the wild-type, this indicates that SPD-2 also plays a role in maintaining SAS-4 after its incorporation into centrioles. In a converse set of experiments, it was found that SPD-2 distribution is not altered in zyg-1(RNAi), sas-5(RNAi), sas-6(RNAi), or sas-4(RNAi) embryos. Overall, it is concluded that SPD-2 controls centriolar recruitment of the four other proteins (Delattre, 2006).

ZYG-1, which is required for the presence of centriolar SAS-5 and SAS-6, which are themselves needed for GFP-SAS-4 recruitment, was investigated. In a converse set of experiments, ZYG-1 distribution was examined in embryos compromised for SAS-5, SAS-6, or SAS-4 function. In the wild-type, levels of ZYG-1 at centrioles are regulated across the cell cycle, with the signal being minimal during interphase and maximal during anaphase. ZYG-1 still localizes to centrioles in sas-5(RNAi) embryos as well as in sas-5(t2079) mutant embryos, in which SAS-5 and SAS-6 are not present at centrioles. ZYG-1 also localizes to centrioles in sas-6(RNAi) and sas-4(RNAi) embryos. Together, these observations establish that ZYG-1 acts upstream of SAS-5 and SAS-6 centriolar recruitment, which themselves act upstream of SAS-4 centriolar recruitment (Delattre, 2006).

In the course of these experiments it was discovered that ZYG-1 levels at centrioles remain high throughout the cell cycle in sas-5(t2079) mutant embryos and sas-6(RNAi) embryos. By contrast, levels of centriolar ZYG-1 still oscillate across the cell cycle in sas-4(RNAi) embryos, with the signal being minimal during interphase and maximal during anaphase. Together, these results indicate that SAS-5 and SAS-6 are required for the diminution of centriolar ZYG-1 during interphase. Because SAS-5 and SAS-6 are present in the cytoplasm but absent from centrioles in sas-5(t2079) mutant embryos, these results suggest in addition that this requirement reflects the presence or activity of centriolar SAS-5 and SAS-6 (Delattre, 2006).

It was of interest to place the recruitment of centriolar microtubules in the pathway that emerges from this study. However, their recruitment could not be assayed using GFP-β-tubulin, because the fusion protein is also incorporated in the remainder of the microtubule cytoskeleton, masking the specific centriolar signal. Therefore, the timing of centriolar microtubule recruitment relative to the five proteins discussed above is not known. Nevertheless, attempts were made to test whether astral microtubules are required for the recruitment of these proteins using RNAi against the alpha-tubulin gene tba-2 (Delattre, 2006).

In severely affected tba-2(RNAi) embryos, tubulin is detected only in paternally contributed centrioles and their immediate vicinity, as expected from the fact that RNAi does not target sperm under these experimental conditions. Interestingly, it was observed that the two paternally contributed centrioles split from one another in one-cell-stage tba-2(RNAi) embryos. Therefore, astral microtubules do not appear to be needed for centriole splitting at the onset of the duplication cycle in C. elegans embryos, as in vertebrate somatic cells. It was noted also that there are only two centrosomes in tba-2(RNAi) embryos, even after several cell cycles. In principle, these two centrosomes could each contain a pair of centrioles if just one round of centriole formation had occurred. However, since centrioles can split from one another in tba-2(RNAi) embryos, four centrosomes, each containing one centriole, would be expected in this scenario. As only two centrosomes are present, it appears instead that completion of daughter centriole formation is impaired and that each centrosome contains only one paternally contributed centriole in tba-2(RNAi) embryos. Therefore, centriole formation does not seem to occur in tba-2(RNAi) embryos. Similarly, centriole formation fails in vertebrate somatic cells treated with high doses of colcemid (Delattre, 2006).

It has been reported that GFP-SAS-5 and GFP-SAS-6 are recruited to centrioles in tba-2(RNAi) embryos. The same is true for GFP-SPD-2, as well as SPD-2, ZYG-1, and GFP-SAS-4. Although the possibility that residual tubulin contributes to the recruitment of these proteins, these results strongly suggest that SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4 can all be recruited independently of astral microtubules (Delattre, 2006).

This study has provided evidence for an emerging pathway for centriole formation in C. elegans. Together with earlier work, these findings lead a proposed sequence of events. Initially, SPD-2 is recruited to each mother centriole or to a closely associated structure. SPD-2 is needed for the centriolar recruitment of ZYG-1, which in turn is required for the remaining three proteins to localize to centrioles. SAS-5 and SAS-6 are recruited next and are needed for SAS-4 to be incorporated thereafter. Furthermore, these results suggest that assembly of centriolar microtubules occurs downstream of SAS-4 incorporation or in parallel to the entire pathway. In addition, the PCM components SPD-5 and gamma-tubulin play a partially redundant role in centriole formation, and it will be interesting to investigate their placement in this sequence. Overall, it is concluded that, like other assembly processes, centriole formation can be described as a series of consecutive steps that entails the sequential recruitment of at least five proteins that ensure formation of a daughter centriole next to each mother centriole once per cell cycle (Delattre, 2006).

SPD-2 is unique among the five proteins investigated in being also required for PCM assembly. In embryos depleted of SPD-2, the coiled-coil protein SPD-5 is not recruited to centrosomes, resulting in the absence of other PCM components, including the Aurora kinase AIR-1 and gamma-tubulin. Thus, SPD-2 lies upstream in the pathway for PCM assembly. Similarly, it was found that SPD-2 is the upstream-most component among the five proteins essential for centriole formation. Therefore, SPD-2 plays a pivotal role in coordinating assembly of the two principal constituents of centrosomes. Perhaps SPD-2 acts in a manner analogous to scaffold proteins in signaling networks, which serve to localize and modulate kinases and their substrates. In this scenario, ZYG-1 and its substrates may be brought together by SPD-2 during centriole formation. spd-2 and zyg-1 exhibit a strong genetic interaction, compatible with the two components having a close relationship. Interestingly, it was discovered that SAS-5 and SAS-are needed for the diminution of centriolar ZYG-1 during interphase. Whereas it remains to be determined whether diminution of centriolar ZYG-1 is important, it is tempting to speculate that this serves as a signal ensuring that SAS-5 and SAS-6 have been recruited before further steps can take place (Delattre, 2006).

Centriole assembly in Caenorhabditis elegans

Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Using electron tomography of staged C. elegans one-cell embryos this study shows that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. Centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms (Pelletier, 2006).

The structure of centrioles is conserved from ancient eukaryotes to mammals. They are barrel-shaped, 100-250 nm in diameter and 100-400 nm in length, with a ninefold symmetric array of singlet, doublet or triplet microtubules. Daughter centrioles grow orthogonally to the older (mother) centrioles, but the assembly process remains mysterious. Centrioles in C. elegans are 150 nm in length, 100 nm in diameter and consist of a central tube surrounded by nine singlet microtubules. Genomic and genetic analyses have identified proteins required for centriole duplication in C. elegans. These include the ZYG-1 kinase (O'Connell, 2001), the SAS proteins (SAS-4, SAS-5 and SAS-6) and SPD-2, all of which localize to centrioles (Pelletier, 2006 and references therein).

C. elegans oocytes arrest in meiotic prophase I, at which point they lack centrioles. Fertilization of oocytes by sperm triggers the completion of meiosis and contributes a centriole pair. After meiosis, the embryo enters the first mitotic division, which is characterized by a series of easily distinguishable events. These include pronuclear appearance (PNA), pronuclear migration (PNM), pronuclear rotation (PNR) and metaphase. Using chemical fixation and serial section electron microscopy it was possible to detect daughter centriole assembly intermediates at, but not before, PNM (Pelletier, 2006).

To determine whether the onset of daughter centriole assembly correlates with the recruitment of known centriole proteins, mating-based assays were performed. In these assays, the sperm donates an unlabelled centriole pair, whereas the green fluorescent protein (GFP)-tagged centriole proteins are contributed by the maternal cytoplasm. Their incorporation into nascent daughter centrioles can therefore be monitored. Embryos were fixed at different stages of the first cell division and stained using SAS-4 antibodies -- to mark the position of the sperm centrioles -- and GFP or ZYG-1 antibodies to monitor the recruitment of individual centriole proteins to the site of assembly. It was observed that SPD-2 and ZYG-1 are recruited to centrioles soon after fertilization, during the completion of female meiosis, whereas SAS-4, SAS-5 and SAS-6 are recruited shortly afterwards, at PNA (Pelletier, 2006).

To gain insights into the assembly relationship between these proteins, individual centriole proteins were depleted by RNA-mediated interference (RNAi) and the effect this had on the recruitment of the other proteins was determined. In one-cell spd-2(RNAi) embryos, no ZYG-1 recruitment was detected at any stage; however, in zyg-1(RNAi) embryos, SPD-2 recruitment was unaffected. These results suggest that SPD-2 acts upstream of ZYG-1. In all spd-2(RNAi) embryos analysed, no significant recruitment of maternal SAS-4, SAS-5 or SAS-6 was detected to the site of centriole assembly. Essentially the same result was observed in zyg-1(RNAi) embryos, which is in agreement with the observation that SPD-2 is required to recruit ZYG-1. In sas-4(RNAi) embryos, both SAS-5 and SAS-6 are recruited to the site of daughter centriole assembly, confirming published results in two-cell embryos. It has been shown that SAS-4 recruitment is blocked in sas-5(RNAi) and sas-6(RNAi) embryos. Together, these results suggest that in one-cell embryos SPD-2 and ZYG-1 are required for daughter centriole assembly by promoting the recruitment of the centriole components SAS-5 and SAS-6. SAS-5 and SAS-6 recruitment then promotes the recruitment of SAS-4 (Pelletier, 2006).

Thin section electron microscopy results showed that daughter centriole assembly begins during PNM. In contrast, mating assays showed that centriole proteins are recruited beforehand, during meiosis or at PNA. Two possibilities could explain these observations. First, centriole proteins may be recruited to the proximity of the mother centrioles before daughter centriole assembly. Second, the recruitment observed may coincide with the emergence of a structural intermediate that is undetectable using chemical fixation and thin section electron microscopy. To examine potential structural intermediates of centriole assembly, an approach was developed to perform high-pressure freezing and electron tomography on isolated embryos at different stages of the first cell division. Surprisingly, daughter centriole assembly intermediates were observed at PNA. The smallest structure detected was a cylindrical tube approximately 60 nm in length, similar to the central tube of mother centrioles. Electron-dense appendages emanated from the singlet microtubules of mother centrioles. During PNM, the central tubes of daughter centrioles were longer, reaching about 110 nm at the end of PNM. The central tube of daughter centrioles increased in diameter during PNM, whereas that of the mother centriole did not; this increase coincided with the appearance of an inner/outer wall structure on daughter centriole central tubes. Singlet microtubules began assembling around the central tube during PNR and by the end of PNR nine singlet microtubules were observed. Hook-like appendages were detected along the length of daughter central tubes at positions where singlet microtubules had yet to assemble. Singlet microtubule assembly did not seem to occur preferentially at distal or proximal extremities, although a slight positional bias for the distal region of the central tube was observed. In summary, tomography results show that in one-cell embryos, daughter centriole assembly begins with the formation of the central tube, which elongates and increases in diameter before the assembly of the nine singlet microtubules (Pelletier, 2006).

The effect was examined of individually depleting centriole proteins on daughter centriole assembly. In zyg-1(RNAi) embryos, no daughter centriole structures were detected, consistent with the idea that in zyg-1(RNAi) embryos, the SAS-proteins are not recruited to the site of centriole assembly. Similarly, in sas-5(RNAi) and sas-6(RNAi) embryos, no daughter centriole structures were observed. Most interestingly, in sas-4(RNAi) embryos at PNA central tubes of daughter centrioles still formed. After PNR, daughter centriole central tubes were longer, suggesting that tube elongation can still occur in sas-4(RNAi) embryos. Daughter centriole central tubes failed to increase in width, and seemed defective in the assembly of both singlet microtubules and hook-like appendages. In older embryos, daughter centriole central tubes were often difficult to discern. These results are consistent with observation for wild-type embryos that the formation of a central tube represents a structural intermediate in centriole assembly, which is unstable in sas-4(RNAi) embryos. Taken together, electron tomography and light microscopy data suggest that SAS-4 is required to assemble or maintain singlet microtubules onto the central tube, the assembly of which is, in turn, dependent on SAS-5 and SAS-6 (Pelletier, 2006).

Using electron tomography this study has shown that daughter centriole assembly in one-cell C. elegans embryos occurs in at least three distinct steps: tube formation; tube elongation; and singlet microtubule assembly (See a model for centriole formation in C. elegans; Delattre, 2006). Assembly begins at the time of PNA and is completed by the end of PNR, which is estimated to require 8-10 min. The recruitment of the SAS-proteins at PNA to the site of daughter centriole assembly is coincident with the emergence of the daughter central tube. In spd-2(RNAi) and zyg-1(RNAi) embryos, the recruitment of the SAS-proteins to the site of assembly is blocked. Accordingly, daughter centriole structures were not detected in zyg-1(RNAi) embryos. These observations, in combination with results showing that SPD-2 is required for the recruitment of the ZYG-1 kinase to the site of daughter centriole assembly, suggest that the role of SPD-2 in centriole duplication is to recruit ZYG-1 to centrioles. ZYG-1 - possibly in concert with SPD-2 - can then either function as a signal required for the initiation of daughter centriole assembly or directly drive the assembly process (Pelletier, 2006).

The fact that daughter centriole central tubes assemble in sas-4(RNAi) embryos (conditions under which SAS-5 and SAS-6 recruitment is not inhibited) indicates that SAS-5 and SAS-6 could be structural components of the central tube. SAS-5 and SAS-6 are known to physically interact, indicating that they may fulfil this structural role as a heterodimer (Leidel, 2005a). Alternatively, it is possible that SAS-5 and SAS-6 are required to maintain the structural integrity of the central tube, which is composed of other proteins. In sas-4(RNAi) embryos, central tube assembly is not perturbed although its stability and capacity to assemble singlet microtubules are compromised. Thus, a role is proposed for SAS-4 in tethering singlet microtubules to the central tube, which would be consistent with the ring-like distribution of SAS-4 around centrioles (Kirkham, 2003). It was previously shown that the amount of SAS-4 on centrioles is related to the quantity of pericentriolar material they recruit (Kirkham, 2003). It is therefore possible that the singlet microtubules on the C. elegans centriole are required to recruit and organize pericentriolar material (Leidel, 2005b). Recent evidence has shown that pericentriolar material is also required for daughter centriole assembly (Dammermann, 2004). It will be of interest to delineate the contribution of such proteins to daughter centriole assembly (Pelletier, 2006).

Published structures using chemical fixation and thin section electron microscopy on mammalian tissue culture cells are ambiguous as to whether centrioles have a central tube. It will be of importance to revisit daughter centriole assembly in mammalian tissue culture cells, and other organisms, using high-pressure freezing and time-resolved tomography to determine if daughter centriole assembly also proceeds in the same order: tube formation, elongation and microtubule assembly. Interestingly, three of the five components required for centriole duplication in C. elegans (SPD-2, SAS-4 and SAS-6) have mammalian homologues, although only human SAS-6 is known to be required for centriole duplication. It is therefore likely that some of the assembly intermediates uncovered here in C. elegans are conserved in mammals and other eukaryotes (Pelletier, 2006).

Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila Centrosomin

Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. This study shows that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles 'rocket' around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and only subtle defects are observed in the asymmetric divisions of mutant neuroblasts. It is concluded that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts (Lucas, 2007).

This study shows that mutations in DSas-4, which encodes the Drosophila homologue of the human microcephaly protein CenpJ/CPAP, also lead to defects in the asymmetric divisions of larval NBs. The defects were much more severe in DSas-4 mutants, which completely lack centrioles/ centrosomes (~15% of NBs divided symmetrically, whereas ~15% failed in cytokinesis). The much milder defects in asymmetric division that were observe in cnn NBs suggest that centrosomes are partially functional as MTOCs in cnn mutant somatic cells. Indeed, relatively well-focused astral MT arrays were frequently observed forming and disassembling in the cytoplasm, and these were often transiently associated with the spindle poles in cnn NBs (Lucas, 2007).

Taken together, these observations on cnn and DSas-4 mutant NBs reveal that, unlike the situation in male GSCs, the asymmetric behavior of the centrosomes is not essential for the accurate asymmetric division of larval NBs. Nevertheless, mutations in the Drosophila homologues of two of the three human centrosomal proteins implicated in microcephaly do lead to relatively subtle defects in NB divisions in flies. Drosophila cnn and DSas-4 mutants do not have small brains, suggesting that flies are able to compensate for defects in these divisions in a way that perhaps humans cannot (Lucas, 2007).

Search PubMed for articles about Drosophila Sas-4

Azimzadeh, J. and Bornens, M. (2007). Structure and duplication of the centrosome. J. Cell. Sci. 120: 2139-2142. Full text of article

Basto, R., Lau, J., Vinogradova, T., Gardiol, A., Woods, C. G., Khodjakov, A. and Raff, J. W. (2006). Flies without centrioles. Cell 125: 1375-1386. Medline abstract: 16814722

Bettencourt-Dias, M. et al. (2005). SAK/PLK4 is required for centriole duplication and flagella development. Curr. Biol. 15: 2199-2207. Medline abstract: 16326102

Bonaccorsi, S., et al. (2000). Spindle assembly in Drosophila neuroblasts and ganglion mother cells. Nat. Cell Biol. 2: 54-56. Medline abstract: 10620808

Bond, J., et al. (2005). A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size. Nat. Genet. 37: 353-355. Medline abstract: 15793586

Dammermann, A., et al. (2004). Centriole assembly requires both centriolar and pericentriolar material proteins. Dev. Cell 7: 815-829. Medline abstract: 15572125

de Anda, F. C., et al. (2005). Centrosome localization determines neuronal polarity. Nature 436: 704-708. Medline abstract: 16079847

Delattre, M., Canard, C. and Gonczy, P. (2006). Sequential protein recruitment in C. elegans centriole formation. Curr. Biol. 16(18): 1844-9. Medline abstract: 16979563

Giansanti, M. G., et al. (2001). The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts. Development 128: 1137-1145. Medline abstract: 11245579

Habedanck, R., et al. (2005). The Polo kinase Plk4 functions in centriole duplication. Nat. Cell Biol. 7: 1140-1146. Medline abstract: 16244668

Kirkham, M., Muller-Reichert, T., Oegema, K., Grill, S. and Hyman, A. A. (2003). SAS-4 is a C. elegans centriolar protein that controls centrosome size. Cell 112: 575-587. Medline abstract: 12600319

Kouprina, N., et al. (2005). The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein, Hum. Mol. Genet. 14: 2155-2165. Medline abstract: 15972725

Leidel, S. and Göczy, P. (2003). SAS-4 is essential for centrosome duplication in C. elegans and is recruited to daughter centrioles once per cell cycle. Dev. Cell 4(3): 431-9. Medline abstract: 12636923

Leidel, S., Delattre, M., Cerutti, L., Baumer, K. and Gonczy, P. (2005a). SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells. Nature Cell Biol. 7: 115-125. Medline abstract: 15665853

Leidel, S. and Gonczy, P. (2005b). Centrosome duplication and nematodes: recent insights from an old relationship. Dev. Cell 9: 317-325. Medline abstract: 16139223

Lucas, E. P. and Raff, J. W. (2007). Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila Centrosomin. J. Cell Biol. 178: 725-732. PubMed citation: 17709428

Martinez-Campos, M., et al. (2004). The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis. J. Cell Biol. 165: 673-683. Medline abstract: 15184400

Megraw, T. L., et al. (2001). Zygotic development without functional mitotic centrosomes. Curr. Biol. 11: 116-120. Medline abstract: 11231128

O'Connell, K. F., et al. (2001). The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo. Cell 105(4): 547-58. Medline abstract: 11371350

Pelletier, L., et al. (2006). Centriole assembly in Caenorhabditis elegans. Nature 444(7119): 619-23. Medline abstract: 17136092

Woods, C. G. et al. Bond, J. and Enard, W. (2005). Autosomal recessive primary microcephaly (MCPH): a review of clinical, molecular, and evolutionary findings. Am. J. Hum. Genet. 76: 717-728. Medline abstract: 15806441

date revised: 30 May 2008

Home page: The Interactive Fly © 2007 Thomas Brody, Ph.D.